The present invention relates to a process for the enzymatic saccharification of a cellulosic substrate to glucose by hydrolyzing it with cellulose enzymes from the microorganism Microbispora bispora, Rutgers P&W (M.b.R.) or a mutant thereof.
Cellulose is said to be the most widely occurring organic compound on earth. It is composed essentially of repeating subunits of D-glucose, linked by .beta.-(1-4)-glycosidic bonds. Total hydrolysis yields D-glucose, and partial hydrolysis gives the disaccharide cellobiose, which is .beta.-D-glucopyranosyl-.beta.-(1-4)-D-glycopyranose. Therefore, cellulose is a .beta.-1,4-glucan.
Cellulose constitutes the major storage form of photosynthesized glucose and the major component of solar energy which has converted to biomass. As worldwide demand for energy and food supplies increases, cellulose in its abundance becomes an attractive raw material for supplying these needs. The glucose subunits of cellulose can be used in a variety of processes for production of energy on the one hand or for use in the production of protein on the other.
A major impediment to cellulose utilization technology, however, has been the difficulty of obtaining glucose in reasonable yield from cellulose while expending reasonable costs in terms of energy input, equipment requirements and the like. Chemical hydrolysis suffers from the drawbacks of high costs of capital equipment, of high processing costs, low yields, production of complex product mixtures and inability to stop the degradation of cellulose at a point which produces primarily the desired product, glucose. Therefore, enzyme-catalyzed saccharification of cellulose is seen as a promising alternative to chemical degradation which can achieve a high efficiency conversion of cellulose to glucose.
Although by tradition, cellulolytic microorganisms are a bane to the pulp and paper industry, causing significant loss through rotting, staining and slime formation, microbial enzymatic decomposition can also be turned to industrial advantage, in the controlled conversion of biomass to ethanol, chemical feedstocks and food. Enzymatic conversion of cellulose to glucose using an enzyme such as cellulase is superior to chemical dissolution in that it proceeds at moderate temperature and pressure, provides recyclable catalysts and frees the environment from the undesirable side products associated with chemical hydrolysis. However, the production of adequate amounts of enzymes such as cellulase is dependent upon identifying a suitable source of substantial quantities of cellulase enzymes in a reasonably pure state.
Cellulase is in actuality a complex of enzymes which act cooperatively, or synergistically, in degrading crystalline cellulose. These enzymes are endo-glucanase, cellobiohydrolase or glucohydrolase, and cellobiase (.beta.-glucosidase). Current thinking is that a cellulosic substrate is initially hydrolyzed by endoglucanases yielding oligomeric intermediates. These oligomeric intermediates are immediately acted upon by exo-splitting glucanases such as glucohydrolase or cellobiohydrolase to produce, respectively, glucose or cellobiose from the non-reducing termini. Both types of glucanases continue to hydrolyze the residual oligomers, and finally cellobiase cleaves the short chain oligomers and cellobiose to yield glucose. It has been found that the most effective cellulases contain both exo- and endo-splitting components, and only those cellulases containing both are able to produce high saccharification conversions of crystalline cellulose. The simultaneous production of both of these types of enzymes by microorganisms appears to be relatively restricted; good yields have been reported to be obtained from only a few fungal genera, including Fusarium, Penicillium, Phanaerochaete (syn. Sporotrichum) and Trichoderma.
The microorganisms of the Trichoderma reesei (T. reesei) species are considered in the art to be the best source of all enzymes in the cellulase complex. However the utility of T. reesei as a cellulase source is hampered by catabolite repression in the synthesis of cellulase; by inactivity of the cellulase at elevated temperatures; and most importantly, by end-product inhibition during saccharification (cellobiose being in T. reesei a strong end-product inhibitor of both endo-glucanase and cellobiohydrolase, with glucose a competitive inhibitor of .beta.-glucosidase).
Accordingly, the isolation and development of a microbial cellulase source wherein the enzyme product is both resistant to end product inhibition and substantially umimpaired at elevated temperature, is of particular industrial interest, and would constitute a significant advance over the art.